The present invention relates to genetically modified cells overexpressing recombinant heparanase, to methods of overexpressing recombinant heparanase in cellular systems and to methods of purifying recombinant heparanase. The invention further relates to nucleic acid constructs for directing the expression of modified heparanase species to which a protease recognition and cleavage sequence has been introduced, to the modified heparanase species expressed therefrom and to their proteolytic products. The invention further relates to in vivo methods of inhibiting heparanase activity.
The extracellular matrix (ECM) acts both as a structural scaffold and as an informational medium. Its dynamic status is determined by cells that secrete both its constituent molecules and enzymes that catalyze the degradation of these molecules. A stasis between ECM degrading enzymes and their inhibitors maintains the integrity of the matrix. While controlled ECM remodeling is fundamental to normal processes, uncontrolled disruption underlies diverse pathological conditions.
Among the integral constituents of basement membrane and ECM are cell adhesion molecules such as laminin and fibronectin, structural components like collagen and ellastin, and proteoglycans including sydecan, serglican, proteoglycan I and II versican (1-2).
For biochemical characterization of a protein and pharmaceutical applications, it is often necessary to overproduce and purify large quantities of the protein. A major consideration when setting up a production scheme for a recombinant protein is whether the product should be expressed intracellularly or if a secretion system can be used to direct the protein to the growth medium. The inherent properties of the protein and the intended applications dictate the expression system of choice. Another consideration when attempting the production of recombinant eukaryotic proteins are the folding and post translational modification processes associated with their natural expression.
Preferably, production is carried out in a cellular system that supports appropriate transcription, translation, and post-translation modification of the protein of interest. Thus, cultured mammalian cells are widely used in applied biotechnology as well as in different disciplines of basic sciences of cellular and molecular biology for producing recombinant proteins of mammalian origin.
One of the most widely used cells for recombinant protein expression, particularly for biotechnological applications, is the Chinese hamster ovary cell line (CHO). Alternatively, baby hamster kidney cells (BHK21), Namalwa cells, Dauidi cells, Raji cells, Human 293 cells, Hela cells, Ehrlich""s ascites cells, Sk-Hep1 cells, MDCK1 cells, MDBK1 cells, Vero cells, Cos cells, CV-1 cells, NIH3T3 cells, L929 cells and BLG cells (mouse melanoma) have also been shown to consecutively express large quantities of recombinant proteins.
These cells are easily transfected with foreign DNA, that can integrate into the host genome to create stable cell lines, with new acquired characteristics (i.e. expression of recombinant proteins). These new cell lines originate from a single cell that has undergone foreign DNA incorporation and are therefore referred to as xe2x80x9ccellular clonesxe2x80x9d.
Since integration of foreign DNA in host cell genome is relatively inefficient, the isolation of cellular clones requires a selection system that discriminates between the stably transformed and the primary cells.
Dihydrofolate reductase deficiency in CHO cells (CHO dhfr-cell line) offers a particularly convenient selection system for cellular clones. Transfection of the dhfr gene along with the gene of interest, results in the survival of clones in a growth medium containing methotrexate (MTX). The higher the number of foreign dhfr gene copies in the cellular clone, the higher the MTX concentration the cells can survive. It has been demonstrated that integration events of foreign DNA into host cell genome often maintain all the components of the transfected DNA. e.g., the selection marker as well as the gene of interest (67).
In contrast to mammalian expression systems, that inherently express limited quantities of recombinant proteins, other expression systems, such as bacteria, yeast, and virus infected insect cells are widely used.
Using such cellular gene expression systems, large amounts of either active or non-active protein can be obtained and used for biochemical analysis of protein properties, structure function relationship, kinetic studies, identification of, screening for, or production of specific inhibitors, production of poly- and monoclonal antibodies recognizing the protein, pharmaceutical applications and the like.
Bacteria are the most powerful tool for the production of recombinant proteins. A recombinant protein that is overproduced in a bacterial system might constitute up to 30% of the total protein content of the cells. The recombinant protein accumulates in inclusion bodies where it is relatively pure (comprises up to 50% of the protein content of the bodies) and protected from protease degradation.
Inclusion bodies enable the accumulation of up to 0.2 grams of protein per liter fermentation culture.
Using specific expression vectors, bacteria can also be directed to produce and secrete proteins into the periplasm and therefrom into the growth medium. Although the reported production quantities are not as high as in inclusion bodies, purification of the expressed protein may be simpler (68).
These advantages and the relative simple growth conditions required for bacteria to thrive, made bacteria a powerful and widely used cellular expression system for the production of recombinant proteins of interest (e.g., human -interferon, human xcex2-interferon, GM-CSF, G-CSF, human LNF-xcex3, IL-2, IL-3, IL-6, TNF, human insulin, human growth hormone, etc.).
Furthermore, non-active bacterialy produced recombinant proteins due to inappropriate folding and disulfide bonding may be reduced and/or denatured and thereafter deoxidized and/or refolded to acquire the catalytically active conformation.
However, when glycosylation of the protein is essential for its activity or uses, eukaryotic expression systems are required.
Yeasts are eukaryotic microorganisms which are widely used for commercial production of recombinant proteins. Examples include the production of insulin, human GM-CSF and hepatitis B antigens (for vaccination) by the yeast Saccharomyces cerevisiae. The relatively simple growth conditions and the fact that yeasts are eukaryotes make the yeast gene expression system highly suitable for the production of recombinant proteins, primarily those with pharmaceutical relevance.
In recent years methylotrophic yeasts (e.g., Pichia pastoris, Hansenula polymorpha) became widely used, thus replacing in many cases the more traditionally used yeast Saccharomyces cerevisiae. 
Methylotrophic yeasts can grow to a high cellular density, and express and if appropriately, secrete, high levels of recombinant proteins. Quantities of the secreted, correctly-folded recombinant protein can accumulate up to several grams per liter culture. These advantages make Pichia pastoris suitable for an efficient production of recombinant proteins (69).
One aspect of the present invention thus concerns the expression of recombinant heparanase in cellular systems.
Heparan Sulfate Proteoglycans (HSPGs)
HSPGs are ubiquitous macromolecules associated with the cell surface and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues (3-7). The basic HSPG structure consists of a protein core to which several linear heparan sulfate chains are covalently attached. The polysaccharide chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are substituted to a varying extent with N- and O-linked sulfate moieties and N-linked acetyl groups (3-7). Studies on the involvement of ECM molecules in cell attachment, growth and differentiation revealed a central role of HSPGs in embryonic morphogenesis, angiogenesis, metastasis, neurite outgrowth and tissue repair (3-7). The heparan sulfate (HS) chains, which are unique in their ability to bind a multitude of proteins, ensure that a wide variety of effector molecules cling to the cell surface (6-8). HSPGs are also prominent components of blood vessels (5). In large vessels they are concentrated mostly in the intima and inner media, whereas in capillaries they are found mainly in the subendothelial basement membrane where they support proliferating and migrating endothelial cells and stabilize the structure of the capillary wall. The ability of HSPGs to interact with ECM macromolecules such as collagen, laminin and fibronectin, and with different attachment sites on plasma membranes suggests a key role for this proteoglycan in the self-assembly and insolubility of ECM components, as well as in cell adhesion and locomotion. Cleavage of HS may therefore result in disassembly of the subendothelial ECM and hence may play a decisive role in extravasation of normal and malignant blood-borne cells (9-11). HS catabolism is observed in inflammation, wound repair, diabetes, and cancer metastasis, suggesting that enzymes which degrade HS play important roles in pathologic processes.
Heparanase
Heparanase is a glycosylated enzyme that is involved in the catabolism of certain glycosaminoglycans. It is an endo-xcex2-glucuronidase that cleaves heparan sulfate at specific intrachain sites (12-15). Interaction of T and B lymphocytes, platelets, granulocytes, macrophages and mast cells with the subendothelial extracellular matrix (ECM) is associated with degradation of heparan sulfate by heparanase activity (16). Connective tissue activating peptide III (CTAP), an -chemokine, was found to have heparanase-like activity. Placenta heparanase acts as an adhesion molecule or as a degradative enzyme depending on the pH of the microenvironvent (17).
Heparanase is released from intracellular compartments (e.g., lysosomes, specific granules) in response to various activation signals (e.g., thrombin, calcium ionophores, immune complexes, antigens and mitogens), suggesting its regulated involvement in inflammation and cellular immunity responses (16).
It was also demonstrated that heparanase can be readily released from human neutrophils by 60 minutes incubation at 4xc2x0 C. in the absence of added stimuli (18).
Gelatinase, another ECM degrading enzyme which is found in tertiary granules of human neutrophils with heparanase, is secreted from the neutrophils in response to phorbol 12-myristate 13-acetate (PMA) treatment (19-20).
In contrast, various tumor cells appear to express and secrete heparanase in a constitutive manner in correlation with their metastatic potential (21).
Degradation of heparan sulfate by heparanase results in the release of heparin-binding growth factors, enzymes and plasma proteins that are sequestered by heparan sulfate in basement membranes, extracellular matrices and cell surfaces (22-23).
Purification of Natural Heparanase
Heparanase activity has been described in a number of cell types including cultured skin fibroblasts, human neutrophils, activated rat T-lymphocytes, normal and neoplastic murine B-lymphocytes, human monocytes and human umbilical vein endothelial cells, SK hepatoma cells, human placenta and human platelets.
A procedure for purification of natural heparanase was reported for SK hepatoma cells and human placenta (U.S. Pat. No. 5,362,641) and for human platelets derived enzymes (62). Purification was performed by a combination of ion exchange and various affinity columns including Con-A Sepharose, Blue A-agarose, Zn++-chelating agarose and Heparin-Sepharose. Evidently, the amounts of active heparanase recovered by these methods is low.
Cloning and Expression of the Heparanase Gene
A purified fraction of heparanase isolated from human hepatoma cells was subjected to tryptic digestion. Peptides were separated by high pressure liquid chromatography (HPLC) and micro sequenced. The sequence of one of the peptides was used to screen data bases for homology to the corresponding back translated DNA sequence. This procedure led to the identification of a clone containing an insert of 1020 base pairs (bp) which included an open reading frame of 963 bp followed by 27 bp of 3xe2x80x2 untranslated region and a poly A tail. The new gene was designated hpa. Cloning of the missing 5xe2x80x2 end of hpa was performed by PCR amplification of DNA from placenta cDNA composite. The joined hpa cDNA (also referred to as phpa) fragment contained an open reading frame which encodes a polypeptide of 543 amino acids with a calculated molecular weight of 61,192 daltons. Cloning an extended 5xe2x80x2 sequence was enabled from the human SK-hep1 cell line by PCR amplification using the Marathon RACE system. The 5xe2x80x2 extended sequence of the SK-hep1 hpa cDNA was assembled with the sequence of the hpa cDNA isolated from human placenta. The assembled sequence contained an open reading frame which encodes a polypeptide of 592 amino acids with a calculated molecular weight of 66,407 daltons. The cloning procedures are described in length in U.S. patent application Ser. Nos. 08/922,170, 09/109,386, and 09/071,618, entitled POLYNUCLEOTIDE ENCODING A POLYPEPTIDE HAVING HEPARANASE ACTIVITY AND EXPRESSION OF SAME IN GENETICALLY MODIFIED CELLS, which is a continuation-in-part of PCT/US98/17954, filed Aug. 31, 1998, all of which are incorporated herein by reference.
The ability of the hpa gene product to catalyze degradation of heparan sulfate (HS) in vitro was examined by expressing the entire open reading frame of hpa in High five and Sf21 insect cells, and the mammalian human 293 embryonic kidney cell line expression systems. Extracts of infected cells were assayed for heparanase catalytic activity. For this purpose, cell lysates were incubated with sulfate labeled, ECM-derived HSPG (peak I), followed by gel filtration analysis (Sepharose 6B) of the reaction mixture. While the substrate alone consisted of high molecular weight material, incubation of the HSPG substrate with lysates of cells infected with hpa containing virus resulted in a complete conversion of the high molecular weight substrate into low molecular weight labeled heparan sulfate degradation fragments (see, for example, U.S. patent application Ser. No. 09/071,618, which is incorporated herein by reference.
In subsequent experiments, the labeled HSPG substrate was incubated with the culture medium of infected High Five and Sf21 cells. Heparanase catalytic activity, reflected by the conversion of the high molecular weight HSPG substrate into low molecular weight HS degradation fragments, was found in the culture medium of cells infected with the pFhpa virus, but not the control pF1 virus.
Altogether, these results indicate that the heparanase enzyme is expressed in an active form by cells infected with Baculovirus or mammalian expression vectors containing the newly identified human hpa gene.
In other experiments, it was demonstrated that the heparanase enzyme expressed by cells infected with the pFhpa virus is capable of degrading HS complexed to other macromolecular constituents (e.g., fibronectin, laminin, collagen) present in a naturally produced intact ECM (see U.S. patent application Ser. No. 09/109,386, which is incorporated herein by reference), in a manner similar to that reported for highly metastatic tumor cells or activated cells of the immune system (7, 8)
Involvement of Heparanase in Tumor Cell Invasion and Metastasis
Circulating tumor cells arrested in the capillary beds often attach at or near the intercellular junctions between adjacent endothelial cells. Such attachment of the metastatic cells is followed by rupture of the junctions, retraction of the endothelial cell borders and migration through the breach in the endothelium toward the exposed underlying base membrane (BM) (24). Once located between endothelial cells and the BM, the invading cells must degrade the subendothelial glycoproteins and proteoglycans of the BM in order to migrate out of the vascular compartment. Several cellular enzymes (e.g., collagenase IV, plasminogen activator, cathepsin B, elastase, etc.) are thought to be involved in degradation of BM (25). Among these enzymes is heparanase that cleaves HS at specific intrachain sites (16,11). Expression of a HS degrading heparanase was found to correlate with the metastatic potential of mouse lymphoma (26), fibrosarcoma and melanoma (21) cells. Moreover, elevated levels of heparanase were detected in sera from metastatic tumor bearing animals and melanoma patients (21) and in tumor biopsies of cancer patients (12).
The inhibitory effect of various non-anticoagulant species of heparin on heparanase was examined in view of their potential use in preventing extravasation of blood-borne cells. Treatment of experimental animals with heparanase inhibitors markedly reduced ( greater than 90%) the incidence of lung metastases induced by B16 melanoma, Lewis lung carcinoma and mammary adenocarcinoma cells (12, 13, 28). Heparin fractions with high and low affinity to anti-thrombin III exhibited a comparable high anti-metastatic activity, indicating that the heparanase inhibiting activity of heparin, rather than its anticoagulant activity, plays a role in the anti-metastatic properties of the polysaccharide (12).
Finally, heparanase externally adhered to B16-F1 melanoma cells increased the level of lung metastases in C57BL mice as compared to control mice (see U.S. patent application Ser. No. 09/260,037, entitled INTRODUCING A BIOLOGICAL MATERIAL INTO A PATIENT, which is a continuation in part of U.S. patent application Ser. No. 09/140,888, and is incorporated herein by reference.
Possible Involvement of Heparanase in Tumor Angiogenesis
Fibroblast growth factors are a family of structurally related polypeptides characterized by high affinity to heparin (29). They are highly mitogenic for vascular endothelial cells and are among the most potent inducers of neovascularization (29-30). Basic fibroblast growth factor (bFGF) has been extracted from a subendothelial ECM produced in vitro (31) and from basement membranes of the cornea (32), suggesting that ECM may serve as a reservoir for bFGF. Immunohistochemical staining revealed the localization of bFGF in basement membranes of diverse tissues and blood vessels (23). Despite the ubiquitous presence of bFGF in normal tissues, endothelial cell proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Studies on the interaction of bFGF with ECM revealed that bFGF binds to HSPG in the ECM and can be released in an active form by HS degrading enzymes (33, 32, 34). It was demonstrated that heparanase activity expressed by platelets, mast cells, neutrophils, and lymphoma cells is involved in release of active bFGF from ECM and basement membranes (35), suggesting that heparanase activity may not only function in cell migration and invasion, but may also elicit an indirect neovascular response. These results suggest that the ECM HSPG provides a natural storage depot for bFGF and possibly other heparin-binding growth promoting factors (36,37). Displacement of bFGF from its storage within basement membranes and ECM may therefore provide a novel mechanism for induction of neovascularization in normal and pathological situations.
Recent studies indicate that heparin and HS are involved in binding of bFGF to high affinity cell surface receptors and in bFGF cell signaling (38, 39). Moreover, the size of HS required for optimal effect was similar to that of HS fragments released by heparanase (40). Similar results were obtained with vascular endothelial cells growth factor (VEGF) (41), suggesting the operation of a dual receptor mechanism involving HS in cell interaction with heparin-binding growth factors. It is therefore proposed that restriction of endothelial cell growth factors in ECM prevents their s systemic action on the vascular endothelium, thus maintaining a very low rate of endothelial cells turnover and vessel growth. On the other hand, release of bFGF from storage in ECM as a complex with HS fragment, may elicit localized endothelial cell proliferation and neovascularization in processes such as wound healing, inflammation and tumor development (36,37).
Recombinant Heparanase for Screening Purposes
Put together, the accumulated evidences indicate that a reliable and high throughput (HTS) screening system for heparanase inhibiting compounds may be applied to identify and develop non-toxic drugs for the treatment of cancer and metastasis. Research aimed at identifying and developing inhibitors of heparanase catalytic activity has been handicapped by the lack of a consistent and constant source of a purified and highly active heparanase enzyme and of a reliable screening system. Such a HTS system is described in U.S. patent application Ser. No. 09/113,168, which is incorporated herein by reference. To this end, however, methods are required for obtaining high quantities of highly pure and active heparanase, so as to enable to study the kinetics of heparanase per se and in the presence of potential inhibitors. The recent cloning, expression and purification of the human heparanase-encoding gene offer, for the first time, a most appropriate and reliable source of active recombinant enzyme for screening of anti-heparanase antibodies and compounds which may inhibit the enzyme and hence be applied to identify and develop drugs that may inhibit tumor metastasis, autoimmune and inflammatory diseases.
Screening for Specific Inhibitors Using a Combinatorial Library
A new approach aimed at rational drug discovery was recently developed for screening for specific biological activities. According to the new approach, a large library of chemically diverged molecules are screened for the desired biological activity. The new approach has become an effective and hence important tool for the discovery of new drugs. The new approach is based on xe2x80x9ccombinatorialxe2x80x9d synthesis of a diverse set of molecules in which several components predicted to be associated with the desired biological activity are systematically varied. The advantage of a combinatorial library over the alternative use of natural extracts for screening for desired biologically active compounds is that all the components comprising the library are known in advance (60).
In combinatorial screening, the number of hits discovered is proportional to the number of molecules tested. This is true even when knowledge concerning the target is unavailable. The large number of compounds, which may reach thousands of compounds tested per day, can only be screened, provided that a suitable assay involving a high throughput screening technique, in which laboratory automation and robotics may be applied, exists.
Expression of Heparanase by Cells of the Immune System
Heparanase catalytic activity correlates with the ability of activated cells of the immune system to leave the circulation and elicit both inflammatory and autoimmune responses. Interaction of platelets, granulocytes, T and B lymphocytes, macrophages and mast cells with the subendothelial ECM is associated with degradation of heparan sulfate (HS) by heparanase catalytic activity (10). The enzyme is released from intracellular compartments (e.g., lysosomes, specific granules) in response to various activation signals (e.g., thrombin, calcium ionophore, immune complexes, antigens, mitogens), suggesting its regulated involvement and presence in inflammatory sites and autoimmune lesions. Heparan sulfate degrading enzymes released by platelets and macrophages are likely to be present in atherosclerotic lesions (42).
Treatment of experimental animals with heparanase alternative substrates (e.g., non-anticoagulant species of low molecular weight heparin) markedly reduced the incidence of experimental autoimmune encephalomyelitis (EAE), adjuvant arthritis and graft rejection (10, 43) in experimental animals, indicating that heparanase inhibitors may be applied to inhibit autoimmune and inflammatory diseases (10,43).
The Involvement of Heparanase in Other Physiological Processes and its Potential Therapeutic Applications
Apart from its involvement in tumor cell metastasis, inflammation and autoimmunity, mammalian heparanase may be applied to modulate bioavailability of heparin-binding growth factors (45); cellular responses to heparin-binding growth factors (e.g., bFGF, VEGF) and cytokines (IL-8) (44, 41); cell interaction with plasma lipoproteins (49); cellular susceptibility to certain viral and some bacterial and protozoa infections (45-47); and disintegration of amyloid plaques (48).
Viral Infection
The presence of heparan sulfate on cell surfaces have been shown to be the principal requirement for the binding of Herpes Simplex (45) and Dengue (46) viruses to cells and for subsequent infection of the cells. Removal of the cell surface heparan sulfate by heparanase may therefore abolish virus infection. In fact, treatment of cells with bacterial heparitinase (degrading heparan sulfate) or heparinase (degrading heparan) reduced the binding of two related animal herpes viruses to cells and rendered the cells at least partially resistant to virus infection (45). There are some indications that the cell surface heparan sulfate is also involved in HIV infection (47).
Neurodegenerative Diseases
Heparan sulfate proteoglycans were identified in the prion protein amyloid plaques of Genstmann-Straussler Syndrome, Creutzfeldt-Jakob disease and Scrape (48). Heparanase may disintegrate these amyloid plaques which are also thought to play a role in the pathogenesis of Alzheimer""s disease.
Restenosis and Atherosclerosis
Proliferation of arterial smooth muscle cells (SMCs) in response to endothelial injury and accumulation of cholesterol rich lipoproteins are basic events in the pathogenesis of atherosclerosis and restenosis (50). Apart from its involvement in SMC proliferation as a low affinity receptor for heparin-binding growth factors, HS is also involved in lipoprotein binding, retention and uptake (51). It was demonstrated that HSPG and lipoprotein lipase participate in a novel catabolic pathway that may allow substantial cellular and interstitial accumulation of cholesterol rich lipoproteins (49). The latter pathway is expected to be highly atherogenic by promoting accumulation of apoB and apoE rich lipoproteins (e.g., LDL, VLDL, chylomicrons), independent of feed back inhibition by the cellular cholesterol content. Removal of SMC HS by heparanase is therefore expected to inhibit both SMC proliferation and lipid accumulation and thus may halt the progression of restenosis and atherosclerosis.
In summary, Heparanase may thus prove useful for conditions such as wound healing, angiogenesis, restenosis, atherosclerosis, inflammation, neurodegenerative diseases and viral infections. Mammalian heparanase can be used to neutralize plasma heparin, as a potential replacement of protamine. Anti-heparanase antibodies may be applied for immunodetection and diagnosis of micrometastases, autoimmune lesions and renal failure in biopsy specimens, plasma samples, and body fluids. Common use in basic research is expected.
ECM Proteases and Their Involvement in Tumor Progression and Metastasis
The cooperation with pericellular proteolysis cascades is required for vascular remodeling during angiogenesis, inflammatory processes, tumor progression and metastasis. In particular, the invasive processes that occur during tumor progressionxe2x80x94local invasion, intravasation, extravasation and metastasis formationxe2x80x94involve extracellular matrix (ECM) degradation by proteases.
Four classes of proteases, are known to correlate with malignant phenotype: (i) cysteine proteases including cathepsin B and L; (ii) aspartyl protease cathepsin D; (iii) serine proteases including plasmin, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), (iv) Matrix metalloproteinases (MMPs) including collagenases, gelatinases A and B (MMP2 and MMP9) and stromelysin (MMP3).
Cathepsins are a family of proteases that are found inside cells in normal physiological conditions. Secretion of cathepsins correlates with various pathological conditions, such as arthritis, Alzheimer""s disease and cancer progression (52).
The lysosomal cystein proteases cthepsin B and L have been suggested to play a role in tumor cell invasion and spread, either by directly cleaving extracellular matrix proteins or indirectly by activating other proteases (53).
Cathepsin B was found to have elevated expression levels in cancer cells. Furthermore, the intracellular distribution of the protein differed between invasive and non-invasive cancer cells. In invasive cells, cathepsin B was found in the plasma membrane, whereas in non-invasive cells it was confined to the lysosomes (56). In human tumor cells cathepsin B was secreted from the cells (53) and was shown to degrade extracellular matrix components (54). Cathepsin B and L have been shown to degrade type IV collagen, laminin and fibronectin in vitro at both acid and neutral pH (54). Both enzymes are able to activate the proenzyme form of the urokinase-type plasminogen activator (pro-uPA), which is secreted by tumor cells and can bind to receptors on the tumor cell surface (55). In this cascade mechanism, the lysosomal cysteine proteases may function as effective mediators of tumor associated proteolysis.
MMPs are a family of zinc dependent endopeptidases. They are secreted as inactive proenzymes and are activated by limited proteolysis (57). During human pregnancy, cytotrophoblasts adopt tumor-like properties: they attach the conceptus to the endometrium by invading the uterus and they initiate blood flow to the placenta by breaching maternal vessels. Matrix metalloproteinase MMP-9 (a type IV collagenase/gelatinase) was shown to be upregulated during cytotrophoblast differentiation along the invasive pathway. Furthermore, it was shown that the activity of that protease specified the ability of the cells to degrade ECM components in vitro (58).
Large body of evidence suggests that the matrix metalloproteinases MMP-2 and MMP-9 play an important role in tumor invasion process (59, 58).
There is clearly a widely recognized need for, and it would be highly advantageous to have, genetically modified cells overexpressing recombinant heparanase or modified species thereof, methods of overexpressing recombinant heparanase in cellular systems and methods of purifying recombinant heparanase, so as to enable, a search for heparanase inhibitors using a high throughput assay and a combinatorial approach.
According to one aspect of the present invention there is provided a recombinant cell comprising a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity, the cell expressing recombinant heparanase.
According to a further aspect of the present invention, there is provided a method of obtaining recombinant heparanase comprising the steps of genetically modifying a cell with an expression vector including a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity, the cell expressing recombinant heparanase.
According to still further features in the described preferred embodiments the polynucleotide sequence is as set forth in SEQ ID NO:1 or a functional part thereof, the part encodes the polypeptide having the heparanase catalytic activity.
According to still further features in the described preferred embodiments the polypeptide includes an amino acid sequence as set forth in SEQ ID NO:2 or a functional part thereof having the heparanase catalytic activity. The functional part may be the result of either genetic engineering natural processing by the transduced cell.
According to still further features in the described preferred embodiments the polynucleotide sequence is selected from the group consisting of double stranded DNA, single stranded DNA and RNA.
According to still further features in the described preferred embodiments the cell is a bacterial cell.
According to still further features in the described preferred embodiments the cell is E. coli. 
According to still further features in the described preferred embodiments the cell is an animal cell.
According to still further features in the described preferred embodiments the animal cell is an insect cell.
According to still further features in the described preferred embodiments the insect cell is selected from the group consisting of High five and Sf21 cells.
According to still further features in the described preferred embodiments the animal cell is a mammalian cell, selected, for example, from the group consisting of a Chinese hamster ovary cell line (CHO), baby hamster kidney cells (BHK21), Namalwa cells, Dauidi cells, Raji cells, Human 293 cells, Hela cells, Ehrlich""s ascites cells, Sk-Hep1 cells, MDCK1 cells, MDBK1 cells, Vero cells, Cos cells, CV-1 cells, NIH3T3 cells, L929 cells and BLG cells (mouse melanoma).
According to still further features in the described preferred embodiments the cell is a yeast cell.
According to still further features in the described preferred embodiments the yeast cell is a methylotrophic yeast.
According to still further features in the described preferred embodiments the yeast cell is selected from the group consisting of Pichia pastoris, Hansenula polymorpha and Saccharomyces cerevisiae. 
According to still further features in the described preferred embodiments the heparanase is human recombinant heparanase.
According to still further features in the described preferred embodiments the polynucleotide sequence is integrated in the cell""s genome rendering the cell a stably transduced.
According to still further features in the described preferred embodiments the polynucleotide sequence is external to the cell""s genome, rendering the cell transiently transduced.
According to still further features in the described preferred embodiments the polynucleotide sequence forms a part of a viral genome infective to the cell, be it bacterial or animal cell.
According to still further features in the described preferred embodiments the polynucleotide sequence encodes, in addition, a signal peptide for protein secretion.
According to still further features in the described preferred embodiments the method further comprising the step of subjecting the cell to a substance which induces secretion into the growth medium of secretable proteins, thereby inducing secretion of the recombinant heparanase into the growth medium.
According to still further features in the described preferred embodiments the substance is selected from the group consisting of thrombin, calcium ionophores, immune complexes, antigens and mitogens.
According to still further features in the described preferred embodiments the calcium ionophore is calcimycin (A23187).
According to still further features in the described preferred embodiments the substance is phorbol 12-myristate 13-acetate (PMA).
According to still further features in the described preferred embodiments the method further comprising the step of purifying the recombinant heparanase.
According to still further features in the described preferred embodiments the purification is effected in part by an ion exchange (e.g., Source-S) column.
According to still further features in the described preferred embodiments the purification is from the cell.
According to still further features in the described preferred embodiments the purification is from a growth medium in which the cell is grown.
According to still further features in the described preferred embodiments the cell is grown in a large biotechnological scale of at least half a liter growth medium.
According to another aspect of the present invention provided is a method of purifying a recombinant heparanase from overexpressing cells or growth medium comprising the steps of adsorbing the recombinant heparanase on an ion exchange (e.g., Source-S) column under low salt conditions, washing the column with low salt solution thereby eluting other proteins, and eluting the recombinant heparanase from the column by a salt gradient or a higher salt concentration.
According to a further aspect of the present invention there is provided a method of activating a heparanase enzyme comprising the step of digesting the heparanase enzyme by a protease.
According to still further features in the described preferred embodiments the protease is selected from the group consisting of a cysteine protease, an aspartyl protease, a serine protease and a meatlloproteinase.
According to still further features in the described preferred embodiments the step of digesting the heparanase enzyme by a protease is effected at a pH in which the protease is active, preferably most active.
According to a further aspect of the present invention there is provided a method of in vivo inhibition of proteolytic processing of heparanase comprising the step of in vivo administering a protease inhibitor.
According to still further features in the described preferred embodiments the protease inhibitor is selected from the group consisting of a cysteine protease inhibitor, an aspartyl protease inhibitor, a serine protease inhibitor and a meatlloproteinase inhibitor.
According to a further aspect of the present invention there is provided a nucleic acid construct comprising a first nucleic acid segment encoding for an upstream portion of heparanase, a second, in frame, nucleic acid sequence encoding a recognition and cleavage sequence of a protease and a third, in frame, nucleic acid sequence encoding for a downstream portion of heparanase, wherein the second nucleic acid sequence is in between the first nucleic acid sequence and the third nucleic acid sequence.
According to still further features in the described preferred embodiments the protease is selected having no recognition and cleavage sequences in the upstream and the downstream portions of heparanase.
According to still further features in the described preferred embodiments the third nucleic acid sequence encodes for a catalytically active heparanase when correctly folded.
According to a further aspect of the present invention there is provided a precursor heparanase protein comprising an upstream portion of heparanase, a mid portion of a recognition and cleavage sequence of a protease and a downstream portion of heparanase, wherein the protease is selected having no recognition and cleavage sequences in the upstream and the downstream portions of heparanase.
According to a further aspect of the present invention there is provided a heparanase protein resulting by digesting the precursor heparanase protein described herein.
According to a further aspect of the present invention there is provided a method of obtaining a homogeneously processed, active heparanase, the method comprising the steps of (a) expressing the precursor heparanase protein in a cell which secretes the precursor heparanase protein into the growth medium to obtain a conditioned growth medium, the precursor heparanase protein including an upstream portion of heparanase, a mid portion of a recognition and cleavage sequence of a protease and a downstream portion of heparanase, wherein the protease is selected having no recognition and cleavage sequences in the upstream and the downstream portions of heparanase; (b) treating the precursor heparanase protein with the protease; and (c) purifying a proteolytic heparanase product having heparanase catalytic activity.
According to a further aspect of the present invention there is provided an antibody comprising an immunoglobulin elicited against recombinant native heparanase.
According to a further aspect of the present invention there is provided an affinity substrate comprising a solid matrix and an immunoglobulin elicited against recombinant native heparanase being immobilized thereto.
According to a further aspect of the present invention there is provided a method of affinity purifying heparanase comprising the steps of (a) loading a heparanase preparation on an affinity substrate including a solid matrix and an immunoglobulin elicited against recombinant native heparanase being immobilized thereto; (b) washing the affinity substrate; and (c) eluting heparanase molecules being adsorbed on the affinity substrate via the immunoglobulin.
The present invention successfully addresses the shortcomings of the presently known configurations by providing cells and methods for expressing recombinant heparanase, methods for purifying the recombinant heparanase and modified heparanase precursor species which can be processed to yield highly active heparanase. Other features and advantages of the various embodiments of the present invention are further addressed hereinunder.